The research proposed is to solubilize, purify and characterize cytochrome P450 from adrenal cortex mitochondria. Together with homogeneous adrenodoxin and adrenodoxin reductase, the purified cytochrome will be used to reconstitute cholesterol side chain cleaving activity. The ability of the reconstituted system to hydroxylate other steroid substrates will also be examined and the differences observed will be explored by altering the method for reconstitution of the components. Those conditions found to be required for optimal cholesterol side chain cleavage will be compared with those found to be necessary for steroid 11Beta-hydroxylation in an attempt to identify unique features and components of each. Since the rate of corticosteroid synthesis is determined by the sidechain cleaving reaction and since it is this reaction which is regulated by ACTH, these studies should permit some increased understanding of the basis for regulation of steroid synthesis in the adrenal. Additional studies using radioimmunoassay will quantify the amounts of each of the components of the mitochondrial hydroxylating pathway in the adrenal and in other steroidogenic tissues. By employing substrate quantities of the purified cytochrome, radioactive cholesterol as substrate and a suitable cytochrome P450 reducing system, a search will be made for sterol intermediates in the conversion of cholesterol to pregnenolone.